Our long-term goal is to facilitate the development of new prevention and therapeutic measures for colon cancer formation by understanding the earliest cellular perturbations that follow APC mutation. In the previous funding periods, we revealed an innovative model wherein APC promotes enterocyte differentiation by controlling retinoic acid biosynthesis. These findings now demonstrate that the functions of APC are not limited to its well-established role in regulating canonical WNT signaling. Mechanistically, we identified the transcriptional co-repressor, C-terminal binding protein (CtBP), as a novel, APC-regulated protein that suppresses retinol dehydrogenases and intestinal cell differentiation in concert with dysregulated LEF-1. Early adenomas taken from FAP patients showed elevated levels of CtBP and LEF1 in comparison to adjacent, uninvolved tissues. Surprisingly, however, these same sections showed little evidence of nuclear ?-catenin indicating that loss of retinoic acid and cell differentiation precedes nuclear accumulation of ?-catenin. We further demonstrated that activation of KRAS promoted the accumulation of nuclear ?-catenin and subsequent intestinal cell proliferation. We also described and uncovered an unexpected connection between APC and a novel DNA demethylase system. This DNA demethylase is upregulated upon loss of APC and retinoic acid and maintains intestinal cells in progenitor-like state. This failed differentiation precedes K-RAS induced proliferation via ?-catenin. It is currently unclear where signals for activating KRAS, and therefore, ?-catenin originate in the context of APC loss. Interestingly, adult zebrafish made deficient in retinoic acid demonstrate both differentiation defects and a profound proliferation response that is coincident with activation of inflammatory stromal cells. Our preliminary findings indicate that adult zebrafish engineered to lack immune cells show intestinal differentiation defects, but no proliferative response when made deficient in retinoic acid. We will now expand our studies to examine whether retinoic acid plays roles in both epithelial cells and in intestinal stromal cells and whether interactions between these two cell populations account for both initiation and progression of colon adenomas. We hypothesize that APC controls the production of retinoic acid. Control of retinoic acid production in intestinal epithelial cells regulates cell potential for differentiation by remodeling the epigenetic landscape. In parallel, retinoic acid acts to suppress intestinal inflammatory responses that are need to stimulate epithelial cell proliferation. This project will expand our understanding of how APC and retinoic acid contribute to intestinal development and differentiation. This understanding could support a testable clinical hypothesis aimed at pharmacological re-activation of retinoid signaling in preventing and treating human colon cancers.